Project Summary/Abstract Recent work establishes that skeletal blood vessels are not just mere channels for blood, but instead play an active role in supporting bone formation. However, it is unknown which types of blood vessels provide this support, how these blood vessels alter the production of bone forming cells from their stem cell progenitors, or how this coupling between blood vessels and bone forming cells is tied to the overall physiologic demand for bone formation. Progress in this area has been impeded by most of the growth factors for blood vessels in bone also having direct effects on bone forming cells, preventing their use to study interactions between blood vessels and bone forming cells. We have recently identified a new growth factor for skeletal blood vessels, SLIT3. SLIT3 is produced by bone forming cells and increases bone formation not by targeting bone forming cells themselves, but instead by promoting outgrowth of blood vessels that in turn support bone formation. Thus, SLIT3 presents an attractive tool to study which blood vessels in bone influence bone formation and how these blood vessels shape the production of bone forming cells. Furthermore, recent advances in understanding skeletal stem cells from ourselves and others identify that bone contains multiple populations of stem cells giving rise to bone forming cells, with each of these stem cells having a different anatomic location and function. It is unlikely that a single type of blood vessel is able to support all of these different of skeletal stem cell populations, and we accordingly hypothesize that different types of skeletal blood vessels each support different types of skeletal stem cells. Here, SLIT3 to both address this hypothesis and establish fundamental paradigms for how endothelial cells support bone formation. In Aim 1, we will study how SLIT3-mediated outgrowth of skeletal blood vessels is driven by signals known to promote bone formation, determining if this SLIT3-driven blood vessel outgrowth is needed for the bone formation response to these signals and also which subsets of bone forming cells are the key producers of SLIT3 needed for these responses. In Aim 2, we will determine which specific bone forming cells are supported by each blood vessel type induced by SLIT3. In Aim 3, we will determine how SLIT3-induced blood vessels shape the differentiation of bone forming cells. Overall, this work will provide a foundation for the development of drugs targeting skeletal blood vessels as a new approach to treat osteoporosis and other skeletal disorders. !